Fluorescence detection of cysteine and homocysteine

ABSTRACT

Embodiments of probes for selectively detecting compounds having a thiol group and an amino group, e.g., cysteine and/or homocysteine, are disclosed, along with methods and kits for detecting the compounds in neutral media with the probes. The probes have a structure according to the general formula where R 1 -R 4  independently are hydrogen hydroxyl, halogen, thiol, thioether, lower aliphatic, or lower alkoxy, x is an integer from 0 to 4, and each R 5  independently is halogen, hydroxyl, thiol, thioether, lower aliphatic, or lower alkoxy. Embodiments of the disclosed probes are capable of undergoing condensation/cyclization reactions with cysteine and/or homocysteine. Cysteine and/or homocysteine can be selectively detected and identified by determining fluorescence emission of the probes at characteristic wavelengths.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/514,697, filed Aug. 3, 2011, which is incorporated by reference inits entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Award RO1 EB002044awarded by National Institutes of Health. The government has certainrights in the invention.

FIELD

Embodiments of fluorescent probes capable of selectively detectingcysteine and homocysteine simultaneously in neutral media are disclosed.

BACKGROUND

Biological thiols are essential in maintaining the appropriate redoxstatus of proteins, cells and organisms (Wood et al., Trends Biochem.Sci. 2003, 28, 32-40; Schultz et al., Eur. J. Biochem. 2000, 267,4904-4911). Cysteine (Cys) is an essential amino acid that is involvedin protein synthesis, detoxification, and metabolism. Elevated levels ofCys have been associated with neurotoxicity (Wang et al., J. Neurosci.2001, 21, 3322-3331). Cys deficiency is involved in slowed growth, hairdepigmentation, edema, lethargy, liver damage, muscle and fat loss, skinlesions, and weakness (Shahrokhian, Anal. Chem. 2001, 73, 5972-5978).Homocysteine (Hcy) has been implicated in various types of vascular andrenal diseases. Elevated Hcy (e.g., >12 μM) in blood is a well-knownrisk factor for cardiovascular, Alzheimer's disease, neutral tubedefects, complications during pregnancy, inflammatory bowel disease, andosteoporosis (Seshadri et al., N. Eng. J. Med. 2002, 346, 476-483;Refsum et al., Annu. Rev. Med. 1998, 49, 31-62). Therefore, thedetermination of Cys and Hcy in vivo is correlated to physiologicalfunctions in diagnosing disease. However, because Cys and Hcy levels areassociated with different diseases despite their similar structures, aneed exists for a method to discriminate between Cys and Hcy.

Significant effort has gone into the development of colorimetric,phosphorescent, and fluorescent probes for these thiol-containing aminoacids to achieve high sensitivity, low cost and ease of detection. Todate, most of the indicators or dosimeters are based on the strongnucleophilicity of the thiol group, and various mechanisms have beenemployed, including Michael addition, cleavage reactions, and others.Though these probes show high sensitivity toward thiol-containingcompounds, the direct detection of Cys (or Hcy) is hampered due tointerference from other thiols.

For example, Cys and Hcy are known to undergo cyclization with aldehydesto form thiazolidines (or thiazinanes). Because both the sulfhydryl andthe amino groups contribute to the cyclization, aldehyde cyclizationenables selectivity for Cys and Hcy over other common thiols such asglutathione (GSH). However, since the aminothiol moieties of Cys and Hcyhave similar reactivities towards aldehydes in general, discriminationof them from each other is challenging using heterocycle formation (Liet al., Chem. Comm. 2009, 5904-5906; Lee et al., Chem. Commun. 2008,6173-6175; Tanaka et al., Chem. Commun. 2004, 1762-1763; Li et al.,Chem. Commun. 2009, 5904-5906; Duan et al., Tetrahedron Lett. 2008, 49,6624-6627; Kim et al., Tetrahedron Lett. 2008, 49, 4879-4881; Zhang etal., Tetrahedron Lett. 2007, 48, 2329-2333; Zhang et al., Org. Lett.2009, 11, 1257-1260; Lim et al., Chem. Commun. 2010, 46, 5707-5709).

SUMMARY

Embodiments of probes capable of selectively detecting thiol-containingcompounds are disclosed. Embodiments of methods for using the probes andkits including the probes also are disclosed.

Some embodiments of the disclosed probe include a fluorophore moietyhaving a chemical structure according to formula I

where R¹-R⁴ independently are hydrogen, hydroxyl, halogen, thiol,thioether, lower aliphatic, or lower alkoxy, x is an integer from 0 to4, and each R⁵ independently is halogen, hydroxyl, thiol, thioether,lower aliphatic, or lower alkoxy, and an α,β-unsaturated carbonylmoiety. In certain embodiments, R¹ is methoxy. In particularembodiments, R¹ is methoxy, R²-R⁴ are hydrogen, and x is 0. In someembodiments, the α,β-unsaturated carbonyl moiety is an acrylate ester.

In certain embodiments, the probe has a chemical structure according togeneral formula II

where R¹-R⁴ independently are hydrogen, hydroxyl, halogen, thiol,thioether, lower aliphatic, or lower alkoxy, x is an integer from 0 to4, and each R⁵ independently is halogen, hydroxyl, thiol, thioether,lower aliphatic, or lower alkoxy. In some embodiments, R¹ is methoxy. Inparticular embodiments, R¹ is methoxy, R²-R⁴ are hydrogen, and x iszero.

Some embodiments of the disclosed probes are capable of undergoing acondensation/cyclization reaction with a compound comprising a thiolgroup and an amino group. Certain embodiments of the disclosed probesproduce a fluorescence spectrum having an emission spectrum maximum at afirst wavelength after condensation with the compound, and subsequentlyproduce a fluorescence spectrum having an emission spectrum maximum at asecond wavelength after cyclization, wherein the first and secondwavelengths are different from one another. In particular embodiments,the probes are capable of undergoing condensation/cyclization reactionswith cysteine and/or homocysteine.

Embodiments of a method for detecting at least one compound having athiol group and an amino group include combining a sample potentiallycomprising at least one compound comprising a thiol group and an aminogroup with a solution comprising a probe having a structure according togeneral formula II, allowing a reaction between the compound and theprobe to proceed for an effective period of time, and detecting the atleast one compound by detecting fluorescence of the solution. In certainembodiments, the solution has a pH of 7-8. In some embodiments, thecompound is cysteine, homocysteine, or a combination thereof.

In some embodiments, fluorescence of the solution is detected byobtaining a fluorescence spectrum after the effective period of time. Incertain embodiments, a fluorescence spectrum of the solution ismonitored over a period of time ranging from zero minutes to a timegreater than or equal to the effective period of time.

In some embodiments, when R¹ in general formula II is methoxy, R² ishydrogen, and x is 0, fluorescence is detected at 377 nm, at 487 nm, orat 377 nm and 487 nm after the effective period of time. In certainembodiments, fluorescence is detected at 377 nm, at 487 nm, or at 377 nmand 487 nm over a period of time ranging from zero minutes to theeffective period of time.

In embodiments where the at least one compound is cysteine, fluorescenceof the solution can be detected at 487 nm after the effective period oftime, e.g., after at least 5 minutes, such as after 5-60 minutes. Inembodiments where the at least one compound is homocysteine,fluorescence of the solution can be detected at 377 nm after theeffective period of time, e.g., after 5-60 minutes.

In some embodiments, the probe solution further includes a surfactant.In certain embodiments, the surfactant is cetyltrimethylammoniumbromide. When a surfactant is included, the effective period of time maybe at least 5 minutes, e.g., 8-10 minutes when R¹ in general formula IIis methoxy, R² is hydrogen, and x is 0. In such embodiments, when the atleast one compound is cysteine, fluorescence of the solution can bedetected at 487 nm after the effective period of time. In someembodiments when the at least one compound is homocysteine, fluorescenceof the solution can be detected at 377 nm 8-10 minutes after combiningthe sample and the solution comprising the probe. In other embodimentswhen the at least one compound is homocysteine, detecting thehomocysteine includes measuring fluorescence of the solution at 377 nmand 487 nm at a first time 8-10 minutes after combining the sample andthe solution comprising the probe, measuring fluorescence of thesolution at 377 nm and 487 nm at a second time after combining thesample and the solution comprising the probe, wherein the second time isgreater than 8-10 minutes, and determining a difference in fluorescenceat each of 377 nm and 487 nm at the first time and the second time,wherein a decrease in fluorescence at 377 nm and a proportional increasein fluorescence at 487 nm indicates presence of homocysteine.

In some embodiments, the at least one compound may be cysteine,homocysteine, glutathione, one or more non-amino thiols, or acombination of any two or more thereof. In such embodiments when theprobe has a formula according to general formula II where R¹ is methoxy,R²-R⁴ are hydrogen, x is 0, and the probe solution includes asurfactant, the effective period of time may be greater than or equal to9 minutes, and the at least one compound is detected by detectingfluorescence of the solution at 377 nm and 487 nm after the effectiveperiod of time. In some embodiments, the at least one compound isfurther identified. Cysteine can be identified based upon stablefluorescence of the solution at 487 nm after 9 minutes and for asubsequent period of time of at least 5 additional minutes, glutathioneand/or non-amino thiols can be identified based upon stable fluorescenceof the solution at 377 nm after 9 minutes and for a subsequent period oftime of at least 5 additional minutes, and homocysteine can beidentified based upon proportionally decreasing fluorescence of thesolution at 377 nm and increasing fluorescence of the solution at 487 nmafter 9 minutes and during a subsequent period of time of at least 5additional minutes.

Embodiments of kits for detecting at least one compound comprising athiol group and an amino group (e.g., cysteine, homocysteine, or acombination thereof) include at least one probe according to generalformula II. In some embodiments, the kit further includes a buffersolution at physiologic pH, such as a phosphate solution at pH 7-8. Incertain embodiments, the buffer solution also includes a surfactant,e.g., cetyltrimethylammonium bromide. Embodiments of the kits mayfurther include a plurality of disposable containers in which a reactionbetween the probe and the at least one compound can be performed. Incertain embodiments, an amount of the probe effective to undergo adetectable change in the probe's fluorescence emission spectrum whenreacted with the at least one compound is premeasured into the pluralityof disposable containers.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the enol-keto tautomerism of2-(2′-hydroxyphenyl)-benzothiazole.

FIG. 2 is a graph of absorbance intensity versus wavelength for thereaction of probe 4 with cysteine over a time period of 0-80 seconds.λ_(ex)=304 nm.

FIG. 3A is a graph of absorbance intensity versus wavelength for thereaction of probe 4 with cysteine over a time period of 0-66 minutes.λ_(ex)=304 nm.

FIG. 3B is a graph of absorbance intensities at 377 nm and 487 nm versustime for the reaction of probe 4 with cysteine over a time period of0-66 minutes. λ_(ex)=304 nm.

FIG. 4 is a graph of absorbance intensity versus wavelength for thereaction of probe 4 with cysteine over a time period of 2.5-66 minutes,illustrating an isoemissive point at 427 nm. λ_(ex)=304 nm.

FIG. 5 is a graph of absorbance intensity versus wavelength illustratingthe time-dependent fluorescence spectral changes of 4 (20 μM) with Hcy(1 equiv) in EtOH:phosphate buffer (20 mM, pH 7.4, 2:8 v/v) over a timeperiod of 0-51 minutes. λ_(ex)=304 nm.

FIG. 6 is a graph of absorbance intensity versus wavelength illustratingthe time-dependent fluorescence spectral changes of 4 (20 μM) with Hcy(1 equiv) in EtOH:phosphate buffer (20 mM, pH 7.4, 2:8 v/v) over a timeperiod of 56-915 minutes. λ_(ex)=304 nm.

FIG. 7A is a graph of absorbance intensity versus wavelength for thereaction of probe 4 with homocysteine over a time period of 0-15 hours.λ_(ex)=304 nm.

FIG. 7B is a graph of absorbance intensities at 377 nm and 487 nm versustime for the reaction of probe 4 with cysteine over a time period of0-1000 minutes. λ_(ex)=304 nm.

FIGS. 8A-D are graphs of absorbance intensity versus wavelength (8A, 8C,8D) or time (10B) illustrating the time-dependent fluorescence spectralchanges of probe 4 (20 μM) with cysteamine (1 equiv) in EtOH:phosphatebuffer (20 mM, pH 7.4, 2:8 v/v). λ_(ex)=304 nm. FIG. 8A illustrates thespectral changes at 0-39 min; FIG. 8B illustrates the time-dependentfluorescence intensity changes at 377 and 487 nm, respectively; FIG. 8Cillustrates the changes in fluorescence at 0-1.5 min; FIG. 8Dillustrates the changes in fluorescence at 2.5-39 min.

FIG. 9 is a graph of absorbance intensity versus wavelength illustratingthe time-dependent fluorescence spectral changes of probe 4 (20 μM) with3-mercaptopropanoic acid (91.2 μM) in EtOH:phosphate buffer (20 mM, pH7.4, 2:8 v/v). λ_(ex)=304 nm. Inset: Time-dependent fluorescenceintensity changes at 377 nm.

FIG. 10 is a graph of absorbance intensity versus wavelengthillustrating the time-dependent fluorescence spectral changes of probe 4(20 μM) with N-acetyl-L-cysteine (20 μM) in EtOH:phosphate buffer (20mM, pH 7.4, 2:8 v/v). λ_(ex)=304 nm. Inset: Time-dependent fluorescenceintensity changes at 377 nm.

FIG. 11 is a series of fluorescence spectra of probe 4 with variousamino acids (Cys, Hcy, leucine, proline, arginine, histidine, valine,methionine, threonine, glutamine, alanine, aspartic acid, norleucine,isoleucine, lysine, cystine and homocystine) after 40 min. λ_(ex)=304nm.

FIG. 12 is a graph of absorbance intensity versus wavelengthillustrating the time-dependent fluorescence spectral changes of probe 4(20 μM) with GSH (1 equiv) in EtOH:phosphate buffer (20 mM, pH 7.4, 2:8v/v). λ_(ex)=304 nm. Inset: Time-dependent fluorescence intensitychanges at 377 nm.

FIG. 13 is a graph of absorbance intensity versus wavelengthillustrating the time-dependent fluorescence spectral changes of probe 4(10 μM) with Cys (2 equiv) in CTAB media (1.0 mM) buffered at 7.4(phosphate buffer, 20 mM). λ_(ex)=304 nm. Inset: time-dependentfluorescence intensity changes at 487 nm.

FIG. 14 is a graph of absorbance intensity versus wavelengthillustrating the time-dependent fluorescence spectral changes of probe 4(10 μM) with Hcy (2 equiv) in CTAB media (1.0 mM) buffered at 7.4(phosphate buffer, 20 mM) over a time period of 0-75 minutes. λ_(ex)=304nm.

FIG. 15 is a graph of absorbance intensity versus time at 377 nm and 487nm illustrating the time-dependent fluorescence spectral changes ofprobe 4 (10 μM) with Hcy (2 equiv) in CTAB media (1.0 mM) buffered at7.4 (phosphate buffer, 20 mM). λ_(ex)=304 nm.

FIG. 16 is a graph of absorbance intensity versus wavelengthillustrating the time-dependent fluorescence spectral changes of probe 4(10 μM) with GSH (2 equiv) in CTAB media (1.0 mM) buffered at 7.4(phosphate buffer, 20 mM). λ_(ex)=304 nm. Inset: time-dependentfluorescence intensity changes at 377 nm.

FIG. 17 is a graph of absorbance intensity versus time at 487 nmillustrating the time-dependent fluorescence intensity changes of probe4 (10 μM) at 487 nm upon adding Cys or Hcy (both 20 μM) in CTAB media(1.0 mM) buffered at 7.4 (phosphate buffer, 20 mM). λ_(ex)=304 nm.

FIG. 18 is a ¹H NMR (400 MHz) spectrum of probe 4 in CDCl₃.

FIG. 19 is a ¹³C NMR (100 MHz) spectrum of probe 4 in CDCl₃.

FIG. 20 is a high-resolution mass spectrum of probe 4 (a2-p 110419191754 #12-20; RT: 0.11-0.19; AV: 9; NL: 7.02E6; T: FTMS+p ESI Full ms[200.00-500.00]).

FIG. 21 is a ¹H NMR (400 MHz) spectrum of HMBT in CDCl₃ obtained fromthe reaction of probe 4 with cysteine.

FIG. 22 is a graph of fluorescence intensity versus wavelengthillustrating the fluorescence spectra (λ_(ex)=304 nm) of probe 4 andHMBT (both 10 μM) in EtOH:phosphate buffer (20 mM, pH 7.4, 2:8 v/v).

FIG. 23 is a ¹H NMR (400 MHz) spectrum of compound 3a in D₂O.

FIG. 24 is a ¹³C NMR (100 MHz) spectrum of compound 3a in D₂O.

FIG. 25 is a high-resolution mass spectrum of compound 3a (5-n21104191745 #8-13; RT: 0.12-0.20; AV: 6; NL 1.30E5; T: FTMS−p ESI Full ms[150.00-600.00]).

FIG. 26 is a ¹H NMR (400 MHz) spectrum of compound 3b in D₂O.

FIG. 27 is a ¹³C NMR (100 MHz) spectrum of compound 3b in D₂O.

FIG. 28 is a ¹H-¹³C COSY NMR spectrum of 3b.

FIG. 29 is a high-resolution mass spectrum of compound 3b(6-n_(—)110419180301 #8-13; RT: 0.12-0.20; AV: 6; NL: 1.32E4; T: FTMS−pESI Full ms [150.00-600.00]).

FIG. 30 is a graph of absorbance versus wavelength illustrating thetime-dependent UV-visible spectral changes of probe 4 (20 μM) with Cys(1 equiv) in EtOH:phosphate buffer (20 mM, pH 7.4, 2:8 v/v).

FIGS. 31A and 31B are graphs of absorbance versus wavelengthillustrating the time-dependent UV-visible spectral changes of probe 4(20 μM) with Hcy (1 equiv) in EtOH:phosphate buffer (20 mM, pH 7.4, 2:8v/v); 0-1 h (FIG. 31A); 75 min-15 h (FIG. 31B).

FIG. 32 is a graph of absorbance versus wavelength illustrating thetime-dependent UV-visible spectral changes of probe 4 (20 μM) withcysteamine (20 μM) in EtOH:phosphate buffer (20 mM, pH 7.4, 2:8 v/v).

FIG. 33 is a graph of absorbance versus wavelength illustrating theUV-visible spectra of probe 4 (20 μM) in the presence and absence of3-mercaptopropanoic acid (91.2 μM) in EtOH:phosphate buffer (20 mM, pH7.4, 2:8 v/v). Reaction time, 1 h.

FIG. 34 is a high-resolution mass spectrum of the conjugate additionadduct of probe 4 with N-acetyl-L-cysteine (9-n2 110419190801 #1-20; RT:0.01-0.31; AV: 20; NL: 1.66E6; T: FTMS−p ESI Full ms [200.00-600.00]).

FIG. 35 is a graph of absorbance versus wavelength illustrating theUV-visible spectra of probe 4 (20 μM) in the presence of differentbiothiols (1 equiv) in EtOH:phosphate buffer (20 mM, pH 7.4, 2:8 v/v).Reaction time, 40 min.

FIG. 36 is a graph of fluorescence intensity versus wavelength for thereaction of probe 4 with varying concentrations of cysteine. λ_(ex)=304nm.

FIG. 37 is a graph of fluorescence intensity versus wavelength for thereaction of probe 4 with varying concentrations of homocysteine.λ_(ex)=304 nm.

FIG. 38A is a graph of fluorescence intensity at 487 nm versus cysteineconcentration for a reaction of probe 4 with cysteine.

FIG. 38B is a graph of fluorescence intensity at 377 nm versushomocysteine concentration for a reaction of probe 4 with homocysteine.

FIG. 39 is a graph of fluorescence intensity versus wavelengthillustrating the fluorescence spectra (λ_(ex)=304 nm) of probe 4 (50 μM)in the presence of cysteine (40 μM) and different concentrations ofhomocysteine (0-12 μM) in EtOH:phosphate buffer (20 mM, pH 7.4, 2:8v/v), reaction time 40 minutes.

FIG. 40 is a graph demonstrating the ratiometric (dual wavelength)response of homocycsteine concentration derived from the data in FIG.39.

FIG. 41 is a graph of fluorescence intensity of probe 4 at 377 nm versushomocysteine concentration derived from the data in FIG. 39.

FIG. 42 is a graph of fluorescence intensity versus wavelengthillustrating the fluorescence spectra (λ_(ex)=304 nm) of probe 4 (50 μM)in the presence of 1) homocysteine (4 μM), 2) homocysteine (4 μM) andcysteine (40 μM), and 3) homocysteine (4 μM), cysteine (40 μM), andglutathione (0.5 μM) in EtOH:phosphate buffer (20 mM, pH 7.4, 2:8 v/v),reaction time 40 minutes.

FIG. 43 is a graph of fluorescence intensity versus wavelengthillustration the fluorescence spectra (λ_(ex)=330 nm) of probe 4 (50 μM)in the presence of varying concentrations of cysteine (0-40 μM) in 10%deproteinized human plasma diluted in EtOH:phosphate buffer (20 mM, pH7.4, 2:8 v/v), reaction time 40 minutes.

FIG. 44 is a graph of fluorescence intensity at 483 nm versus cysteineconcentration derived from the data in FIG. 43.

FIG. 45 is a graph of fluorescence intensity versus wavelengthillustration the fluorescence spectra (λ_(ex)=330 nm) of probe 4 (50 μM)in the presence of varying concentrations of homocysteine (0-12 μM) andcysteine (40 μM) in 10% deproteinized human plasma diluted inEtOH:phosphate buffer (20 mM, pH 7.4, 2:8 v/v), reaction time 40minutes.

FIG. 46 is a graph demonstrating the ratiometric (dual wavelength)response of homocycsteine concentration in the presence of excesscysteine and 10% deproteinized human plasma (derived from the data inFIG. 45).

FIG. 47 is a graph of fluorescence intensity of probe 4 at 378 nm versushomocysteine concentration derived from the data in FIG. 45.

DETAILED DESCRIPTION

Simultaneous determination of cysteine (Cys) and/or homocysteine (Hcy)via a single probe remains a significant challenge due to the structuralsimilarity of Cys and Hcy which differ by a single methylene unit intheir side chains.

Disclosed herein are embodiments of probes that can differentiatebetween Cys and Hcy at physiologic pH based on their relativelydifferent intramolecular cyclization rates. Embodiments of the disclosedprobes also can discriminate Cys and Hcy from other amino acids andthiols at physiologic pH. Embodiments of the disclosed probes include afluorophore moiety to facilitate detection and a moiety capable ofundergoing a condensation-cyclization reaction (referred to as a“cyclization moiety”) with Cys and/or Hcy. In some embodiments, thecyclization moiety is an α,β-unsaturated carbonyl moiety, which iscovalently bonded to the fluorophore moiety.

I. TERMS AND DEFINITIONS

The following explanations of terms and abbreviations are provided tobetter describe the present disclosure and to guide those of ordinaryskill in the art in the practice of the present disclosure. As usedherein, “comprising” means “including” and the singular forms “a” or“an” or “the” include plural references unless the context clearlydictates otherwise. The term “or” refers to a single element of statedalternative elements or a combination of two or more elements, unlessthe context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. Other features of thedisclosure are apparent from the following detailed description and theclaims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, percentages, temperatures, times, and soforth, as used in the specification or claims are to be understood asbeing modified by the term “about.” Accordingly, unless otherwiseindicated, implicitly or explicitly, the numerical parameters set forthare approximations that may depend on the desired properties soughtand/or limits of detection under standard test conditions/methods. Whendirectly and explicitly distinguishing embodiments from discussed priorart, the embodiment numbers are not approximates unless the word “about”is recited.

Definitions of common terms in chemistry may be found in Richard J.Lewis, Sr. (ed.), Hawley's Condensed Chemical Dictionary, published byJohn Wiley & Sons, Inc., 1997 (ISBN 0-471-29205-2).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Absorbance: The retention by a compound or substance of certainwavelengths of radiation incident upon it; a measure of the amount oflight at a particular wavelength absorbed as the light passes through acompound or substance, or through a solution of a compound or substance.

Aliphatic refers to a substantially hydrocarbon-based compound, or aradical thereof (e.g., C₆H₁₃, for a hexane radical), including alkanes,alkenes, alkynes, including cyclic versions thereof, and furtherincluding straight- and branched-chain arrangements, and all stereo andposition isomers as well. Unless expressly stated otherwise, analiphatic group contains from one to twenty-five carbon atoms; forexample, from one to fifteen, from one to ten, from one to six, or fromone to four carbon atoms. The term “lower aliphatic” refers to analiphatic group containing from one to ten carbon atoms. An aliphaticchain may be substituted or unsubstituted. Unless expressly referred toas an “unsubstituted aliphatic,” an aliphatic groups can either beunsubstituted or substituted. An aliphatic group can be substituted withone or more substituents (up to two substituents for each methylenecarbon in an aliphatic chain, or up to one substituent for each carbonof a —C═C— double bond in an aliphatic chain, or up to one substituentfor a carbon of a terminal methine group). Exemplary aliphaticsubstituents include, for instance, amine, amide, sulfonamide, halogen,cyano, carboxy, hydroxy, mercapto, trifluoromethyl, alkyl, alkoxy,alkylthio, thioalkoxy, arylalkyl, heteroaryl, alkylamino, dialkylamino,or other functionality.

Alkyl refers to a hydrocarbon group having a saturated carbon chain. Thechain may be branched or unbranched. The term lower alkyl means thechain includes 1-10 carbon atoms.

Alkoxy refers to a functional group having the formula —OR where R is analkyl group. The term lower alkoxy means that the alkyl group includes1-10 carbon atoms.

An analogue or derivative is a compound that is derived from a similarcompound, or a compound that can be imagined to arise from anothercompound, for example, if one atom is replaced with another atom orgroup of atoms. Analogues may differ from one another in one or moreatoms, functional groups, or substructures, which are replaced withother atoms, groups, or substructures.

Aromatic or aryl compounds typically are unsaturated, cyclichydrocarbons having alternate single and double bonds. Benzene, a6-carbon ring containing three double bonds, is a typical aromaticcompound.

Cyclization moiety: As used herein, the term “cyclization moiety” refersto a portion of a molecule capable of undergoing acondensation-cyclization reaction with a target compound, such as acompound comprising a thiol group and an amino group.

Detect: To determine if an agent (such as a target molecule) is presentor absent, for example, in a sample. “Detecting” refers to any method ofdetermining if something exists, or does not exist, such as determiningif a target molecule is present in a biological sample. For example,“detecting” can include using a visual or a mechanical device todetermine if a sample displays a specific characteristic.

Effective period of time: As defined herein, an effective period of timeis a sufficient amount of time to allow a chemical reaction to occur.With respect to the present disclosure, an effective period of time isan amount of time sufficient to allow condensation of a thiol-containingcompound with an embodiment of the disclosed probes and/or subsequentcyclization of the thiol-containing compound to occur.

Emission or emission signal: The light of a particular wavelengthgenerated from a source. In particular examples, an emission signal isemitted from a fluorophore after the fluorophore absorbs light at itsexcitation wavelength(s).

Fluorescence is the emission of visible radiation by an atom or moleculepassing from a higher to a lower electronic state, wherein the timeinterval between absorption and emission of energy is 10⁻⁸ to 10⁻³second. Fluorescence occurs when the atom or molecule absorbs energyfrom an excitation source (e.g., an ultraviolet lamp) and then emits theenergy as visible radiation. The term “stable fluorescence intensity”refers to a fluorescence intensity that remains substantially the sameover a period of time. Substantially the same means that thefluorescence intensity changes by less than 20%, less than 15%, lessthan 10%, less than 5%, or less than 2% over a defined period of time.

A fluorophore, or fluorogen, is a compound capable of fluorescence, suchas a fluorescent dye. The term “fluorophore” also refers to the portionof a molecule that causes the molecule to fluoresce when exposed to anexcitation source.

A functional group is a specific group of atoms within a molecule thatis responsible for the characteristic chemical reactions of themolecule. Exemplary functional groups include, without limitation,alkane, alkene, alkyne, arene, halo (fluoro, chloro, bromo, iodo),epoxide, hydroxyl, carbonyl (ketone), aldehyde, carbonate ester,carboxylate, ether, ester, peroxy, hydroperoxy, carboxamide, amine(primary, secondary, tertiary), ammonium, imide, azide, cyanate,isocyanate, thiocyanate, nitrate, nitrite, nitrile, nitroalkane,nitroso, pyridyl, phosphate, sulfonyl, sulfide, thiol (sulfhydryl),disulfide.

Heteroaryl compounds are aromatic compounds having at least oneheteroatom, i.e., one or more carbon atoms in the ring has been replacedwith an atom having at least one lone pair of electrons, typicallynitrogen, oxygen, or sulfur.

An isoemissive point is a wavelength, wavenumber, or frequency at whichthe total intensity of emission of light by a sample does not changeduring a chemical reaction of physical change.

As used herein, the term “probe” refers to a molecule capable ofselectively reacting with a molecule of interest (i.e., a molecule forwhich the presence and/or concentration is to be determined) andproducing a detectable signal or change as a result of the reaction. Adetectable signal or change may include a change in the absorbancespectrum and/or emission spectrum of the probe and/or the molecule ofinterest.

A substituent is an atom or group of atoms that replaces another atom ina molecule as the result of a reaction. The term “substituent” typicallyrefers to an atom or group of atoms that replaces a hydrogen atom on aparent hydrocarbon chain or ring.

Substituted: A fundamental compound, such as an aryl or aliphaticcompound, or a radical thereof, having coupled thereto, typically inplace of a hydrogen atom, a second substituent. For example, substitutedaryl compounds or substituents may have an aliphatic group coupled tothe closed ring of the aryl base, such as with toluene. Again solely byway of example and without limitation, a long-chain hydrocarbon may havea substituent bonded thereto, such as one or more halogens, an arylgroup, a cyclic group, a heteroaryl group or a heterocyclic group.

Surfactant: A compound that reduces surface tension when dissolved inwater or aqueous solutions. Surfactants typically are amphiphilicorganic compounds, i.e., organic compounds that contain both hydrophobicgroups and hydrophilic groups. Surfactants may be characterized by theirhydrophilic groups, or heads. A non-ionic surfactant includes no formalcharge in its head. Ionic surfactants include hydrophilic groups havinga net charge. If the charge is negative, the surfactant is an anionicsurfactant. If the charge is positive, it is a cationic surfactant. Ifthe head contains two oppositely charged groups, it is a zwitterionicsurfactant.

II. OVERVIEW OF REPRESENTATIVE EMBODIMENTS

Embodiments of probes that can differentiate between cysteine andhomocysteine at physiologic pH are disclosed. Also disclosed areembodiments of methods and kits for performing the detection.

Embodiments of the disclosed probes include (a) a fluorophore moietyhaving a chemical structure according to formula I

where R¹-R⁴ independently are hydrogen, hydroxyl, halogen, thiol,thioether, lower aliphatic, or lower alkoxy, x is an integer from 0 to4, and each R⁵ independently is halogen, hydroxyl, thiol, thioether,lower aliphatic, or lower alkoxy; and (b) an α,β-unsaturated carbonylmoiety. In one embodiment, R¹ is methoxy. In another embodiment, R¹ ismethoxy, R²-R⁴ are hydrogen, and x is 0. In any or all of the aboveembodiments, the α,β-unsaturated carbonyl moiety may be an acrylateester.

In some embodiments, the probe has a chemical structure according toformula II

where R¹-R⁴ and x are as defined above. In one embodiment, R¹ ismethoxy. In another embodiment, R¹ is methoxy, R²-R⁴ are hydrogen, and xis 0.

In any or all of the above embodiments, the probe may be capable ofundergoing a condensation/cyclization reaction with a compoundcomprising a thiol group and an amino group. In some embodiments, theprobe has a first fluorescence spectrum having an emission spectrummaximum at a first wavelength after condensation with the compound, andthe probe has a subsequent fluorescence spectrum having an emissionspectrum maximum at a second wavelength after cyclization, wherein thefirst and second wavelengths are different from one another. In any orall of the above embodiments, the compound may be cysteine,homocysteine, or a combination thereof.

Embodiments of a method for detecting at least one compound comprising athiol group and an amino group include combining a sample potentiallycomprising at least one compound comprising a thiol group and an aminogroup with a solution comprising a probe having a structure according togeneral formula II, allowing a reaction between the compound and theprobe to proceed for an effective period of time, and detecting the atleast one compound by detecting fluorescence of the solution. In someembodiments, the solution has a pH of 7-8. In any or all of the aboveembodiments, the at least one compound may be cysteine, homocysteine, ora combination thereof.

In any or all of the above embodiments, fluorescence of the solution maybe detected by obtaining a fluorescence spectrum after the effectiveperiod of time, or by monitoring a fluorescence spectrum of the solutionover a period of time ranging from zero minutes to a time greater thanor equal to the effective period of time.

In any or all of the above embodiments, the probe may have a structureaccording to general formula II where R¹ is methoxy, R²-R⁴ are hydrogen,and x is 0. In one such embodiment, detecting fluorescence of thesolution includes detecting the fluorescence at 377 nm, at 487 nm, or at377 nm and 487 nm after the effective period of time. In another suchembodiment, detecting fluorescence of the solution includes detectingthe fluorescence at 377 nm, at 487 nm, or at 377 nm and 487 nm over aperiod of time ranging from zero minutes to the effective period oftime. In some embodiments, the at least one compound is cysteine anddetecting the at least one compound comprises detecting fluorescence ofthe solution at 487 nm after the effective period of time; the effectiveperiod of time may be at least 5 minutes, such as 5-60 minutes. In otherembodiments, the at least one compound is homocysteine and detecting theat least one compound comprises detecting fluorescence of the solutionat 377 nm after the effective period of time; the effective period oftime may be 5-60 minutes.

In any or all of the above embodiments, the solution may further includea surfactant. The surfactant may be cetyltrimethylammonium bromide. Insome embodiments, the effective period of time is at least 5 minutes. Insome embodiments, the probe has a structure according to general formulaII where R¹ is methoxy, R² is hydrogen, and x is 0. In such embodiments,the effective period of time may be 8-10 minutes. In one embodiment, theat least one compound is cysteine, and detecting the cysteine includesdetecting fluorescence of the solution at 487 nm after the effectiveperiod of time. In another embodiment, the at least one compound ishomocysteine, and detecting the homocysteine includes detectingfluorescence of the solution at 377 nm 8-10 minutes after combining thesample and the solution comprising the probe. In still anotherembodiment, the at least one compound is homocysteine, and detecting thehomocysteine includes (1) measuring fluorescence of the solution at 377nm and 487 nm at a first time 8-10 minutes after combining the sampleand the solution comprising the probe; (2) measuring fluorescence of thesolution at 377 nm and 487 nm at a second time after combining thesample and the solution comprising the probe, wherein the second time isgreater than 8-10 minutes; and (3) determining a difference influorescence at each of 377 nm and 487 nm at the first time and thesecond time, wherein a decrease in fluorescence at 377 nm and aproportional increase in fluorescence at 487 nm indicates presence ofhomocysteine. In yet another embodiment, the at least one compoundcomprises cysteine, homocysteine, glutathione, one or more non-aminothiols, or a combination thereof, the effective period of time isgreater than or equal to 9 minutes, and detecting the at least onecompound comprises detecting fluorescence of the solution at 377 nm and487 nm after the effective period of time. The method may furtherinclude identifying the at least one compound, wherein cysteine isidentified based upon stable fluorescence intensity of the solution at487 nm after 9 minutes and for a subsequent period of time of at least 5additional minutes, glutathione and/or non-amino thiols are identifiedbased upon stable fluorescence intensity of the solution at 377 nm after9 minutes and for a subsequent period of time of at least 5 additionalminutes, and/or homocysteine is identified based upon proportionallydecreasing fluorescence intensity of the solution at 377 nm andincreasing fluorescence intensity of the solution at 487 nm after 9minutes and during a subsequent period of time of at least 5 additionalminutes.

Embodiments of a kit for detecting at least one compound comprising athiol group and an amino group include at least one probe according togeneral formula II. The at least one compound may be cysteine,homocysteine, or a combination thereof. In any or all of the aboveembodiments, the kit may further include a buffer solution atphysiologic pH. In some embodiments, the buffer solution is a phosphatesolution at pH 7-8. The buffer solution may further include asurfactant, such as cetyltrimethylammonium bromide. In any or all of theabove embodiments, the kit may also include a plurality of disposablecontainers in which a reaction between the probe and the at least onecompound can be performed. In some embodiments, an amount of the probeeffective to undergo a detectable change in the probe's fluorescenceemission spectrum when reacted with the at least one compound ispremeasured into the plurality of disposable containers.

III. Overview of Conjugate Addition-Cyclization

Cysteine (Cys) and homocysteine (Hcy) are structurally related,differing in the presence of a single extra methylene group in the sidechain of Hcy. Cysteine is capable of undergoing condensation withacrylates to form substituted 1,4-thiazepines (Blondeau et al., Can. J.Chem. 1971, 49, 3866-3876; Leonard et al., J. Org. Chem. 1966, 31,3928-3935). The reaction involves the conjugate addition of Cys toacrylates (1) to generate thioether (2), which can further undergo anintramolecular cyclization to yield the desired compound 3a(3-carboxy-5-oxoperhydro-1,4-thiazepine), as illustrated in Scheme 1.

An analogous thioether can be generated from Hcy (Khatik, Org. Lett.2006, 8, 2433-2436). However, the intramolecular cyclization reaction toform an eight-membered ring is kinetically disfavored relative to theseven-membered ring as in the case of Cys, i.e., homocysteine'scyclization rate is expected to be less than cysteine's cyclizationrate.

IV. PROBES

Embodiments of the disclosed probes include a fluorophore moiety and acyclization moiety capable of undergoing a condensation-cyclizationreaction with a compound comprising a thiol group and an amino group. Incertain embodiments, the cyclization moiety is an α,β-unsaturatedcarbonyl moiety. In some embodiments, the probe is capable of undergoinga condensation/cyclization reaction with Cys and/or Hcy. In certainembodiments, the α,β-unsaturated carbonyl moiety is capable ofundergoing condensation/cyclization with both Cys and Hcy, but withmeasurably different reaction rates. In particular embodiments, theprobe is capable of producing an emission spectrum maximum at a firstwavelength after condensation with a thiol-containing compound, and theprobe further is capable of producing an emission spectrum maximum at asecond wavelength after condensation with a compound comprising a thiolgroup and an amino group and subsequent cyclization of the compound withcleavage and release of the fluorophore moiety, wherein the first andsecond wavelengths are not the same. In general, fluorophores that formO-acyl or N-acyl groups when conjugated to the α,β-unsaturated carbonylmoiety may be suitable fluorophore moieties.

One suitable fluorophore is 2-(2′-hydroxyphenyl)benzothiazole (HBT)(FIG. 1). As shown in FIG. 1, HBT can undergo an excited-stateintramolecular photon transfer (ESIPT) process upon photo excitationwhereby rapid photoinduced proton transfer results in tautomerizationbetween its enol and keto forms. The enol form produces fluorescentemission at a short wavelength, and the keto form produces fluorescentemission at a long wavelength. Accordingly, HBT exhibits dual emissionbands, which originate from its enol and keto tautomeric forms(Lochbrunner et al., J. Chem. Phys. 2000, 112, 10699-10702). Modifyingthe hydroxyl group of HBT blocks ESIPT, resulting exclusively inenol-like emission. If the free hydroxyl group is regenerated,tautomerization resumes and dual emission bands reappear.

The inventors hypothesized that masking the hydroxyl group of HBT withan α,β-unsaturated carbonyl moiety capable of undergoing acondensation/cyclization reaction with Cys and/or Hcy might generate aprobe capable of distinguishing between Cys and Hcy, wherein the probewould be capable of dual fluorescence emission (upon excitation with alight source) after the α,β-unsaturated carbonyl moiety was removed bythe condensation/cyclization reaction.

The probe would emit little or no fluorescence prior to condensationwith the thiol group. After condensation, the probe would emitfluorescence at the wavelength corresponding to the enol form, and thefluorescence from the keto form would increase over time as the reactionprogressed with cyclization of Cys and/or Hcy and concomitant release ofthe fluorophore moiety. The fluorescence from the keto form would beexpected to increase more slowly in the presence of Hcy than in thepresence of Cys due to the difference in reaction rates. Although theprobe may be capable of condensation with non-amino-containing thiolsand/or thiols with a secondary or tertiary amino group e.g.,glutathione, cyclization and release of the fluorophore is unlikely tooccur.

In some embodiments, the probe comprises a fluorophore moiety derivedfrom HBT or a derivative thereof wherein the fluorophore moiety has achemical structure according to formula I

where R¹-R⁴ independently are hydrogen, hydroxyl, halogen, thiol,thioether, lower aliphatic, or lower alkoxy, x is an integer from 0 to4, and each R⁵ independently is halogen, hydroxyl, thiol, thioether,lower aliphatic, or lower alkoxy. In some embodiments, upon cleavage ofthe cyclization moiety from the fluorophore moiety, a fluorophorecomprising an electron donating group (e.g., hydroxyl, thiol, thioether,lower alkoxy) in a position ortho or meta to C¹ in general formula I isreleased. In certain embodiments, R¹ is methoxy. In particularembodiments, R¹ is methoxy, R²-R⁴ are hydrogen, and x is 0.

Suitable HBT derivatives include substitutedhydroxyphenylbenzothiazoles. In certain embodiments, the fluorophoremoiety is derived from 2-(2′-hydroxy-3′-methoxyphenyl)-benzothiazole(HMBT). HMBT has fluorescence emission bands at 377 nm (enol form) and487 nm (keto form). In at least some embodiments, the presence of a 3′methoxy group (i.e., R¹=methoxy) provided stronger fluorescence at 377nm than a corresponding probe where R¹ and R² were hydrogen.

The probe further comprises a cyclization moiety attached whereindicated in general formula I. In some embodiments, the cyclizationmoiety is an α,β-unsaturated carbonyl moiety. In certain embodiments,the α,β-unsaturated carbonyl moiety is a substituted or unsubstitutedacrylate ester moiety capable of reacting with a thiol group, e.g., athiol group on Cys or Hcy. Suitable substituents may include halogen,hydroxyl, lower alkoxy, lower aliphatic, or aryl groups. In oneembodiment, an acrylate ester substituted with a benzene ring on the αcarbon (structure A) reacted poorly with thiol-containing compounds anddemonstrated almost no response, perhaps due to steric hindrance fromthe benzene ring.

Thus, in certain embodiments, the acrylate ester is unsubstituted orincludes substituents unlikely to produce steric hindrance, e.g.,halogen, hydroxyl, lower alkoxy (such as methoxy, ethoxy) or loweraliphatic (such as methyl, ethyl, propyl) groups. In particularembodiments, the acrylate ester is unsubstituted and has the formulaH₂C═CHC(O)O—.

In some embodiments, the probe has a structure according to generalformula II

where R¹-R⁴ independently are hydrogen, hydroxyl, halogen, thiol,thioether, lower aliphatic, or lower alkoxy, x is an integer from 0 to4, and each R⁵ independently is halogen, hydroxyl, thiol, thioether,lower aliphatic, or lower alkoxy. In certain embodiments, R¹ is methoxy.In one embodiment, R¹ is methoxy, R²-R⁴ are hydrogen, and x is zero(i.e., probe 4).

V. PROBE SYNTHESIS

In some embodiments, a probe is synthesized using HBT or HMBT as thefluorophore moiety. HMBT can be synthesized by reacting2-aminothiophenol with o-vanillin in ethanol. An α,β-unsaturatedcarbonyl moiety is added by acylating the free hydroxyl group on thefluorophore with an acrylic acid derivative, e.g., acryloyl chloride.The synthesized probes typically are only weakly fluorescent due toquenching of the fluorophore by the carbon-carbon double bond via aphoto-induced electron transfer (PET) process.

VI. CYSTEINE AND HOMOCYSTEINE DETECTION AND DIFFERENTIATION

Embodiments of the disclosed probes are capable of undergoingcondensation and cyclization with at least some compounds that include athiol group and an amino group. In some embodiments, the probes arecapable of undergoing condensation/cyclization with compounds thatinclude a terminal thiol group and a primary amino group, e.g., cysteineand/or homocysteine. Some embodiments of the disclosed probes may becapable of undergoing a condensation reaction without subsequentcyclization with other thiol-containing compounds, such as thiolcompounds that include no amino group and/or thiol compounds thatinclude a secondary or tertiary amino group.

Probe 4 reacts with Cys (n=1) and Hcy (n=2) as shown in Scheme 2.

As shown in Scheme 2, the acrylate ester moiety of probe 4 combines withthe thiol group in cysteine and homocysteine to form compounds 5a and5b, respectively. Subsequent cyclization of Cys and Hcy with theacrylate ester moiety and concomitant elimination of HMBT producescyclic compounds 3a and 3b, respectively.

In some embodiments, a probe according to general formula II is combinedin solution with a sample potentially including at least one compoundhaving a thiol group and an amino group. A reaction between the compoundand the probe is allowed to proceed for an effective period of time. Insome embodiments, the reaction is performed at a temperature rangingfrom 0° C. to 95° C., such as 10° C. to 60° C., 15° C. to 50° C., or 20°C. to 30° C. In certain embodiments, the reaction is performed atambient temperature. An effective period of time is an amount of timesufficient to allow condensation and/or cyclization to occur. Theeffective period of time may depend, at least in part, on the reactiontemperature. In some embodiments, an effective period of time is atleast 5 minutes at least 7 minutes, at least 9 minutes, or at least 10minutes. In certain embodiments, effective periods of time range from 5minutes to 20 hours, such as 5 minutes to 15 hours, 5 minutes to 5hours, 5 minutes to 2 hours, 5-75 minutes, 5-60 minutes, 5-40 minutes,or 9-60 minutes. In some embodiments, the reaction is performed atphysiologic pH, e.g., a pH of 7-8, such as a pH of 7.3-7.5. In certainembodiments, the reaction is performed at pH 7.4.

After the effective period of time, the at least one compound can bedetected by fluorescence. In some embodiments, a fluorescence spectrumover a range of wavelengths, e.g., 200-600 nm is obtained after theeffective period of time. In certain embodiments, a plurality offluorescence spectra are obtained over a period of time ranging fromzero minutes to a time greater than or equal to the effective period oftime. For example, a plurality of spectra may be obtained over a timeperiod ranging from zero minutes to 20 hours, 0 minutes to 15 hours, 0minutes to 2 hours, 0-75 minutes, 0-60 minutes, 0-40 minutes, 5-75minutes, 5-60 minutes, 5-40 minutes, or 5-30 minutes.

In some embodiments, fluorescence is measured or monitored at one ormore wavelengths corresponding to expected emission spectrum maxima ofthe probe. For example, when the probe is probe 4, fluorescence may bemeasured or monitored at 377 nm (corresponding to the enol form of probe4) and/or 487 nm (corresponding to the keto form of probe 4). Iffluorescence at 377 nm is detected after the effective period of time,it indicates that the solution includes at least one compound capable ofundergoing condensation with probe 4. If fluorescence at 487 nm isdetected after the effective period of time, it indicates that thesolution includes at least one compound capable of undergoingcondensation with probe 4 and subsequent cyclization with release of thefluorophore moiety.

In a working embodiment, fluorescence evaluation of the reaction betweenprobe 4 and Cys demonstrated that emission at 377 nm increases initiallydue to conjugate addition which removes the alkene-induced PET quenching(FIG. 2). Upon further reaction, the emission band at 377 nmsuccessively decreases with concomitant growth of the keto band at 487nm (FIGS. 3A-B). A well-defined isoemissive point appears at 427 nm(FIG. 4). The latter spectral change is due to lactam formation whichresults in the formation of HMBT exhibiting ESIPT.

In the case of Hcy, the conjugate addition reaction leads to thioether3b. However, the rate for subsequent eight-membered ring lactamformation 3b is relatively slow compared to the formation of 3a. In aworking embodiment, emission at 377 nm steadily increased over time for55 minutes (FIG. 5), followed by a decrease in emission at 377 nm after55 minutes accompanied by an increase of the emission at 487 nm (FIG. 6,FIGS. 7A-B).

Control experiments demonstrate that the amino group of Cys is neededfor the selective cyclization reaction. When cysteamine was reacted withprobe 4, similar fluorescence changes are observed as for Cys underanalogous reaction conditions (FIGS. 8A-D). However, 3-mercaptopropanoicacid (MPA) exhibits fluorescence emission centered at 377 nm due to theformation of the conjugate addition product only (FIG. 9).N-acetyl-L-cysteine (NAC) produces a similar result to that of MPA,formation of the conjugate addition adduct (FIG. 10). Thus, the aminogroup is involved in the intramolecular cyclization reaction, and thereaction does not occur in the absence of the amino group.

Probe selectivity for Cys and Hcy was demonstrated by evaluating changesin fluorescence intensity of 4 caused by other analytes, such asleucine, proline, arginine, histidine, valine, methionine, threonine,glutamine, alanine, aspartic acid, norleucine, isoleucine, lysine,cystine and homocystine. As shown in FIG. 11, only Cys and Hcy exhibitedsignificant fluorescence intensity changes at 487 and 377 nm,respectively, after 40 minutes while other amino acids caused nofluorescence intensity changes under the same conditions.

GSH can also react with embodiments of the disclosed probes to produceenol-like emission due to the conjugate addition reaction (see, e.g.,FIG. 12), potentially interfering with Cys and/or Hcy detection.However, GSH and other sulfhydryls are not capable of cyclizing andreleasing the fluorophore; thus, GSH and other sulfhydryls do notproduce fluorescence emission at 487 nm when reacted with probe 4, butmay produce fluorescence emission at 377 nm. Interference from GSH andother sulfhydryls can be overcome by addition of a surfactant thatincreases the cyclization and release rates of Cys and Hcy. In someembodiments, the surfactant is a cationic surfactant, such as aquaternary ammonium surfactant. In one embodiment, the surfactant wascetyltrimethylammonium bromide (CTAB). Surfactants such as CTAB increasethe reaction rate of Cys and Hcy with probe 4, resulting in a more rapidincrease in fluorescence emission at 487 nm (FIGS. 13-16). As shown inFIG. 17, the formation of HMBT from probe 4 and Cys is complete within 9minutes. In the case of Hcy, almost no free HMBT emission can beobserved in 9 minutes (FIG. 17); however, HMBT emission at 487 nmincreases over time, allowing Hcy to be distinguished from GSH and othersulfhydryls that are incapable of cyclization with release of thefluorophore.

The detection limits of Cys and Hcy are 0.11 μM and 0.18 μM,respectively, which is below the requisite detection limits for Cys andHcy in human plasma. The assay can distinguish concentration changes onthe order of 2-3 μM. Such sensitivity enables sensitivity distinguish,for instance, normal (5-12 μM) Hcy levels, hyperhomocysteinemia (16-100μM, indicating cardiovascular risk) and homocysteinuria (>100 μM, asevere inherited metabolic disorder associated with mental retardation,a multisystemic disorder of the connective tissues, muscles, centralnervous system, and cardiovascular system).

Thus, in some embodiments of the disclosed method, a solution comprisinga probe according to general formula II and a surfactant is combinedwith a sample potentially including at least one compound having a thiolgroup and an amino group. The reaction is allowed to progress for aneffective period of time, and the at least one compound is detected bydetecting fluorescence of the solution. In certain embodiments, thesurfactant is cetyltrimethylammonium bromide. In particular embodiments,R is methoxy. When R is methoxy, fluorescence can be monitored at 377 nmand/or 487 nm to detect the at least one compound. If the at least onecompound is cysteine, fluorescence can be detected at 487 nm after theeffective period of time. In some embodiments, when the at least onecompound is homocysteine, fluorescence can be detected at 377 nmsubstantially immediately after the effective period of time, e.g., at8-10 minutes after combining the sample and the probe solution. Incertain embodiments when the at least one compound is homocysteine,fluorescence of the solution is measured at 377 nm and 487 nmsubstantially immediately after the effective period of time and againat a second, later time. The difference in the fluorescence measurementsat the first and second times at each wavelength is determined. Ifhomocysteine is present, a decrease in fluorescence at 377 nm and aproportional increase in fluorescence at 487 nm will be observed.

In some embodiments, the sample may further include glutathione and/ornon-amino thiols. Glutathione and other non-amino thiols may undergocondensation with the probe, but are unable to subsequently cyclize andrelease the fluorophore. Thus, in such embodiments, Cys can be measuredvia a stable signal (i.e., stable fluorescence intensity) appearing at487 nm substantially immediately after the effective period of time,e.g., after 8-10 minutes after (indicating rapid condensation andcyclization). As used herein with respect to fluorescence intensity, theterm “stable” means that the fluorescence intensity changes by less than20%, such as less than 15%, less than 10%, less than 5% or less than 2%over a subsequent period of time. In some embodiments, the fluorescenceintensity remains stable for a subsequent period of time of at least 5additional minutes, at least 10 additional minutes, at least 15additional minutes, at least 30 additional minutes, or at least 45additional minutes, such as 5-60 additional minutes, 5-55 additionalminutes, 10-50 additional minutes, or 15-45 additional minutes followingthe effective period of time. GSH and non-amino thiols can be measuredvia a stable signal at 377 nm after the effective period of time(indicating condensation but no cyclization). Hcy is the only analytethat causes a proportional change of the signals at 377 nm and 487 nmafter the effective period of time as it slowly cyclizes with release ofthe fluorophore. Accordingly, Hcy can be detected by monitoringfluorescence over time because the respective signals due to Cys (487nm) and other sulfhydryls (377 nm) stabilize after 9 minutes.

VII. KITS

Kits are also a feature of this disclosure. Embodiments of the kitsinclude at least one probe according to general formula II, wherein theprobe is suitable for selectively detecting one or more thiol-containingcompounds, particularly one of more compounds including a thiol groupand an amino group (e.g., cysteine, homocysteine). In certainembodiments, R in general formula II is methoxy. In some embodiments,the kit further includes a buffer solution at physiologic pH. In certainembodiments, the buffer is a phosphate buffer at pH 7-8, such as a 20 mMphosphate buffer at pH 7.4. The probe may be dissolved in the buffer, orthe probe may be included in a dry form and the user can combine theprobe and the buffer solution at or before the time of use. In someembodiments, the buffer solution further includes a surfactant, e.g.,cetyltrimethylammonium bromide. The kits also may include one or morecontainers, such as a disposable test tube or cuvette, in which thedetection can be performed. In some embodiments, an amount of the probeeffective to undergo a detectable change in the fluorescence emissionspectrum, or both when reacted with the at least one compound ispremeasured into the disposable containers. The kits may further includeinstructions for performing the detection. In some embodiments, the kitsinclude control samples of thiol-containing compounds, e.g., cysteineand/or homocysteine. Typically the control samples are provided in solidform.

VIII. EXAMPLES Materials and Instruments

All chemicals were purchased from Sigma-Aldrich and Acros and usedwithout further purification. ¹H-NMR and ¹³C-NMR spectra were recordedon a Bruker AMX-400 NMR spectrometer, using TMS as an internal standard.ESI-HRMS (high resolution mass spectrometry) spectra were obtained on aThermo Electron LTQ Orbitrap hybrid mass spectrometer. UV-visiblespectra were collected on a Cary 50 UV-Vis spectrophotometer.Fluorescence spectra were collected on a Cary Eclipse (Varian, Inc.)fluorescence spectrophotometer with slit widths set at 5 and 10 nm forexcitation and emission, respectively. The pH measurements were carriedout with an Orion 410A pH meter. In all experiments enantiomeric allypure natural amino acids were used except for homocysteine, which wasused as the racemate.

Example 1 Synthesis of Probe 4

2-(2′-hydroxy-3′-methoxyphenyl)benzothiazole (HMBT)

HMBT was synthesized according to a previous reported method with minormodifications (Guo et al., Chin. Chem. Lett. 2009, 20, 1408-1410. Asolution of 2-aminothiophenol (0.3 mL, 4.2 mmol) and o-vanillin (0.48 g,3.15 mmol) in EtOH (10 mL), aq H₂O₂ (30%, 18.9 mmol) and aq HCl (37%,9.45 mmol) was stirred at room temperature for 90 min. The solution wasquenched by 10 mL H₂O. The precipitate was filtered, dried under vacuumand recrystallized from EtOH to afford the desired product as a lightyellow solid (0.64 g, 79% yield). ¹H NMR (CDCl₃, 400 MHz), δ (ppm):12.75 (s, 1H), 8.01 (d, 1H, J=7.6 Hz), 7.91 (d, 1H, J=7.2 Hz), 7.51 (t,1H, J=6.8 Hz), 7.42 (t, 1H, J=7.2 Hz), 7.33 (dd, 1H, J₁=1.2 Hz, J₂=8.0Hz), 6.99 (dd, 1H, J₁=1.2 Hz, J₂=8.0 Hz), 6.91 (t, 1H, J=8.0 Hz), 3.96(s, 1H). ¹³C NMR (CDCl₃, 100 MHz), δ 169,74, 151.86, 149.13, 148.29,136.47, 132.70, 126.80, 125.66, 122.36, 121.59, 120.11, 119.23, 116.86,114.21, 56.36. ESI-FTMS m/z=258.0587 [M+H]⁺, calc. 258.0589 forC₁₄H₁₂NO₂S.

Probe 4

To a solution of HMBT (129 mg, 0.5 mmol) and Et₃N (2 eq) in 10 mL ofanhydrous CH₂Cl₂, acryloyl chloride (1.25 eq, mixed with 4 mL of CH₂Cl₂)was added dropwise at 0° C. After stirring at this temperature 90 min,the mixture was warmed to room temperature and stirred overnight. Thesolution was diluted with CH₂Cl₂ (30 mL), washed with H₂O (15 mL×3) anddried over anhydrous Na₂SO₄. The solvent was removed in vacuo to furnisha crude mixture (dark brown oil) which afforded 4 upon crystallizationfrom hexanes/CHCl₃ (20:1, v/v) as a light orange solid (110 mg, 71%yield). ¹H NMR (CDCl₃, 400 MHz), δ (ppm) (FIG. 18): 8.08 (d, 1H, J=8.0Hz), 7.99 (dd, 1H, J₁=1.6 Hz, J₂=8.0 Hz), 7.90 (d, 1H, J=8.0 Hz), 7.49(t, 1H, J=7.2 Hz), 7.41-7.34 (m, 2H), 7.11 (dd, 1H, J₁=1.2 Hz, J₂=8.4Hz), 6.74 (dd, 1H, J₁=1.2 Hz, J₂=17.6 Hz), 6.52 (m, 1H), 6.14 (d, 1H,J₁=1.2 Hz, J₂=10.4 Hz), 3.89 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz) (FIG.19), δ 163.50, 162.24, 152.83, 138.10, 135.74, 133.52, 127.77, 127.62,126.91, 126.36, 125.82, 125.41, 123.52, 121.46, 121.42, 114.21, 56.45.ESI-FTMS (FIG. 20) m/z=312.0705 [M+H]⁺, calc. 312.0694 for C₁₇H₁₄NO₃S.

Example 2 Condensation and Cyclization of Cysteine and Homocysteine withProbe 4

To a 100 mL flask, probe 4 (0.12 g, 3.85 mmol) and Cys (1.25 eq) werecombined in 30 mL of MeOH:H₂O (90:10, v/v) solution, and the mixturestirred at room temperature for 1 hour. Triethylamine (Et₃N) (80 μL) wasadded, and the solution was stirred for about 40 minutes. The solventwas dried in vacuo, and the crude product was subjected to columnchromatography (eluted with CH₂Cl₂:MeOH, 10:4, v/v) to afford 85 mg ofHMBT and 49 mg of 3a as an off-white solid.

To a 100 mL flask, probe 4 (0.12 g, 3.85 mmol) and Hcy (1.25 eq) werecombined in 30 mL of MeOH—H₂O (90:10, v/v) solution, and the mixture wasstirred at room temperature for 4 hours. Et₃N (80 μL) was added and thesolution stirred at room temperature overnight. The solvent was dried invacuo and the crude product was subjected to column chromatography(eluted with CH₂Cl₂:MeOH, 10:6, v/v) to afford 78.2 mg of HMBT and 37.7mg of 3b as an off-white solid.

Spectroscopy confirmed the reaction mechanism. Formation of HMBT wasdemonstrated by comparison of the product's ¹H NMR data with that ofauthentic HMBT (FIG. 21). FIG. 22 is a comparison of the fluorescencespectra (λ_(ex)=304 nm) of probe 4 and HMBT (both 10 μM) inEtOH:phosphate buffer (20 mM, pH 7.4, 2:8 v/v). The formation ofcompounds 3a and 3b was confirmed by ¹H NMR, ¹³C NMR, ¹H-¹³C COSY NMRand high-resolution mass spectroscopy (HRMS) (FIGS. 23-29). The spectraclearly demonstrated that an intramolecular cyclization is involved inthe selective signaling event.

3a: ¹H NMR (CDCl₃, 400 MHz), δ (ppm) (FIG. 23): 4.34 (dd, 1H, J₁=2.0 Hz,J₂=9.2 Hz), 3.10-3.00 (m, 2H), 2.93-2.75 (m, 4H). ¹³C NMR (CDCl₃, 100MHz) (FIG. 24), δ 178.64, 175.17, 59.92, 39.61, 33.67, 135.53, 23.03.ESI-FTMS (FIG. 25) m/z=174.0228 [M−H]⁻, calc. 174.0225 for C₆H₈NO₃S.

3b: ¹H NMR (D₂O, 400 MHz), δ (ppm) (FIG. 26): 4.61 (dd, 1H, J₁=3.6 Hz,J₂=12.4 Hz), 3.07-2.90 (m, 3H), 2.78-2.62 (m, 2H), 2.43-2.35 (m, 1H),2.23-2.14 (m, 1H), 1.96 (t, 1H, J=12 Hz). ¹³C NMR (CDCl₃, 100 MHz) (FIG.27), δ 177.94, 177.42, 55.92, 36.66, 30.82, 29.85. From its structure,compound 3b is predicted to have 7 peaks in its ¹³C spectrum. However,only 6 peaks were observed. The peak at 36.66 can be attributed to twooverlapping carbons. A ¹H-¹³C COSY NMR spectrum of 3b confirmed thisanalysis (FIG. 28). ESI-FTMS (FIG. 29) m/z=188.0381 [M−H]⁻, calc.188.0381 for C₇H₁₀NO₃S.

The fluorescence sensing behavior of probe 4 toward Cys was investigatedusing a 20 μM solution of probe 4 in EtOH:H₂O (2:8, v/v) solutionbuffered at pH 7.4 (phosphate buffer, 0.01 M). Upon addition of Cys (1equiv), the emission at 377 nm increased initially (FIG. 2). As thereaction progressed, the emission band at 377 nm successively decreasedwith concomitant growth of the keto band at 487 nm (FIGS. 3A-B, and awell-defined isoemissive point appeared at 427 nm (FIG. 4). FIG. 30depicts the UV-visible spectral changes as the reaction progressed over65 minutes.

In the case of Hcy, emission at 377 nm steadily increased over time for55 minutes (FIG. 5), followed by a decrease in emission at 377 nm after55 minutes accompanied by an increase of the emission at 487 nm (FIG. 6,FIGS. 7A-B). FIGS. 31A-B depict the UV-visible spectral changes as thereaction progressed over 65 minutes.

Example 3 Probe 4 Selectivity Characterization

Control experiments were carried out to prove that the amino group ofCys was needed in the selective cyclization reaction. First, cysteamine(1 equivalent) was combined with probe 4 (20 μM) in ethanol:phosphatebuffer (20 mM, pH 7.4, 2.8 v/v). Fluorescence was evaluated over timeusing an excitation wavelength of 304 nm. Similar fluorescence changeswere observed as for Cys under analogous reaction conditions (FIGS.8A-D). FIG. 32 depicts the UV-visible spectral changes as the reactionprogressed over 40 minutes.

Next, 3-mercaptopropanoic acid (MPA, 91.2 μM) was combined with probe 4in ethanol:phosphate buffer (20 mM, pH 7.4, 2.8 v/v). Fluorescence wasevaluated over time using an excitation wavelength of 304 nm (FIG. 9).MPA exhibited fluorescence emission centered at 377 nm, demonstratingthat only the conjugate addition product formed. FIG. 33 depicts theUV-visible spectra of probe 4 in the presence and absence of MPA after 1hour.

N-acetyl-L-cysteine (NAC, 20 μM) was combined with probe 4 inethanol:phosphate buffer (20 mM, pH 7.4, 2.8 v/v). Fluorescence wasevaluated over time using an excitation wavelength of 304 nm (FIG. 10).NAC produced a similar result to that of MPA, i.e., formation of theconjugate addition adduct. The latter product was evidenced via HRMSdata (ESI-FTMS m/z=473.0850 [M−H]⁻, calc. 473.0841 for C₂₂H₂₁N₂O₆S₂,FIG. 34).

Probe selectivity for Cys and Hcy was demonstrated by evaluating changesin fluorescence intensity of 4 caused by other analytes, such asleucine, proline, arginine, histidine, valine, methionine, threonine,glutamine, alanine, aspartic acid, norleucine, isoleucine, lysine,cystine and homocystine. Each analyte (10 μM) was combined with probe 4(20 μM) in ethanol:phosphate buffer (20 mM, pH 7.4, 2.8 v/v).Fluorescence was evaluated after 40 minutes using an excitationwavelength of 304 nm. As shown in FIG. 11, only Cys and Hcy exhibitedsignificant fluorescence intensity changes at 487 and 377 nm,respectively, while other amino acids caused no fluorescence intensitychanges under the same conditions.

Glutathione (GSH), however, was discovered to produce enol-like emissionat 377 nm. GSH (1 equivalent) was combined with probe 4 inethanol:phosphate buffer (20 mM, pH 7.4, 2.8 v/v). Fluorescence wasevaluated over 255 minutes using an excitation wavelength of 304 nm(FIG. 12). As shown in FIG. 12, fluorescence at 377 nm increased formore than 50 minutes before leveling off. FIG. 35 depicts the UV-visiblespectra of probe 4 in the presence Cys, Hcy, and GSH after 40 minutes.

Analogous cyclocondensation reactions of aminothiols are catalyzed bysurfactants (Sharma et al., Tetrahedron Lett. 2008, 49, 4269-4271).Thus, a surfactant-containing media was prepared with 10 mMcetyltrimethylammonium bromide (CTAB) buffered to pH 7.4 in phosphatebuffer (20 mM). Reaction of Cys (2 equivalents) in the CTAB media wasevaluated over 114 minutes. Fluorescence intensity at 487 nm rapidlyincreased, and was complete within 9 minutes (FIG. 13). Reaction of Hcy(2 equivalents) in the CTAB media was evaluated over 75 minutes. Asshown in FIGS. 14 and 15, fluorescence intensity at 377 nm initiallyincreased for about 15 minutes, and then decreased; fluorescenceintensity at 487 nm slowly increased over time in a linear fashion.Reaction of GSH (2 equivalents) in the CTAB media was evaluated over 6minutes (FIG. 16). Fluorescence intensity at 377 nm increased veryrapidly and began to level off after 3 minutes. The reaction appeared tobe complete after about 5 minutes. No significant fluorescence at 487 nmwas evident.

FIG. 17 demonstrates that Cys and Hcy can be differentiated by theiremission at 487 nm in CTAB media. Fluorescence emission from Cys reacheda maximum within 9 minutes and levels off. In contrast, very littlefluorescence emission from Hcy was seen at 9 minutes, and emissioncontinued to increase over a reaction time of 75 minutes.

Example 4 Simultaneous Determination of Cysteine and Homocysteine

The kinetic differences in the intramolecular cyclization reactions of5a and 5b (Scheme 2) observed in Example 3, indicated that Cys and Hcycould be simultaneously determined over a time course. Cys and Hcy werecombined separately with probe 4 in EtOH:phosphate buffer (0.01 M, pH7.4) (2:8, v/v), and fluorescence was measured over 40 minutes. As shownin FIGS. 36 and 37, fluorescence intensity at 487 nm increased withincreasing Cys concentration (FIB. 36) and fluorescence intensity at 377nm increased with increasing Hcy concentration (FIG. 37). Thefluorescent intensity was linearly proportional to the amount of Cysfrom 0 to 20 μM (FIG. 38A) and 0 to 25 μM for Hcy (FIG. 38B). Thedetection limits of Cys and Hcy are 0.11 μM and 0.18 μM, respectively,which is below the requisite detection limits for Cys and Hcy assays inhuman plasma samples. The assay can distinguish concentration changes onthe order of 2-3 μM. Such

Cysteine and homocysteine were determined simultaneously in a singlesolution. A solution including Cys (40 μM) and Hcy (0-12 μM) was addedto a solution of probe 4 (50 μM) in ethanol:phosphate buffer (20 mM, pH7.4, 2:8 v/v) at ambient temperature. Fluorescence intensity (λ_(ex)=304nm) was measured 40 minutes after the solutions were combined. As shownin FIG. 39, Hcy was detectable at 377 nm with no significantinterference from a physiological concentration of cysteine. As expectedfluorescence intensity at 377 nm increased as the concentration of Hcyincreased. The ratio of fluorescence intensity at 377 nm to fluorescenceintensity at 487 nm increased linearly as Hcy concentration increased(FIG. 40). FIG. 41 demonstrates that fluorescence intensity at 377 nmincreased linearly as Hcy concentration increased.

Homocysteine also can be determined in the presence of both cysteine andglutathione. Solutions of 1) Hcy (4 μM), 2) Hcy (4 μM) and Cys (40 μM),or 3) Hcy (4 μM), Cys (40 μM), and GSH (0.5 μM) were combined with probe4 (50 μM) in ethanol:phosphate buffer (20 mM, pH 7.4, 2:8 v/v) atambient temperature. Fluorescence intensity (λ_(ex)=304 nm) was measured40 minutes after the solutions were combined. FIG. 42 demonstrates thatHcy was detectable with no significant interference from physiologicalconcentrations (i.e., plasma level concentrations) of glutathione,providing further evidence that probe 4 has potential utility inclinical diagnosis.

Example 5 Simultaneous Determination of Cysteine and Homocysteine inDeproteinized Human Plasma

Further evaluation demonstrated that cysteine can be detected indeproteinized human plasma using probe 4. Cysteine and homocysteine alsowere detected simultaneously in deproteinized human plasma with probe 4.

Probe 4 (50 μM) was combined with cysteine (0-40 μM) in 10%deproteinized human plasma diluted in ethanol:phosphate buffer (20 mM,pH 7.4, 2:8 v/v) at ambient temperature. Fluorescence intensity(λ_(ex)=330 nm) was measured 40 minutes after the solutions werecombined. To reduce the background fluorescence from human plasma, theexcitation wavelength was selected at 330 nm instead of maximumexcitation wavelength 304 nm. FIG. 43 demonstrates that fluorescenceintensity at 475-500 nm increased as Cys concentration increased.Fluorescence intensity at 483 nm increased linearly as Cys concentrationincreased (FIG. 44), with reaction times similar to those observed inbuffer.

Next, probe 4 (50 μM) was combined with Hcy (0-12 μM) and Cys (40 μM) in10% deproteinized human plasma diluted in ethanol:phosphate buffer (20mM, pH 7.4, 2:8 v/v) at ambient temperature. Fluorescence intensity(λ_(ex)=330 nm) was measured 40 minutes after the solutions werecombined. As shown in FIG. 45, fluorescence intensity at 375-380 nmincreased as Hcy concentration increased. The ratio of fluorescenceintensity at 378 nm to fluorescence intensity at 483 nm increasedlinearly as Hcy concentration increased (FIG. 46). FIG. 47 demonstratesthat fluorescence intensity at 378 nm increased linearly as Hcyconcentration increased. Moreover, the fluorescence increase at 378 nmcan be observed for Hcy even in the presence of excess Cys.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A probe, comprising: a fluorophore moiety having a chemical structure according to formula I

where R¹-R⁴ independently are hydrogen, hydroxyl, halogen, thiol, thioether, lower aliphatic, or lower alkoxy, x is an integer from 0 to 4, and each R⁵ independently is halogen, hydroxyl, thiol, thioether, lower aliphatic, or lower alkoxy; and an α,β-unsaturated carbonyl moiety.
 2. The probe of claim 1 where R¹ is methoxy.
 3. The probe of claim 1 where R¹ is methoxy, R²-R⁴ are hydrogen, and x is
 0. 4. The probe of claim 1 where the α,β-unsaturated carbonyl moiety is an acrylate ester.
 5. The probe of claim 1 where the probe has a chemical structure according to formula II


6. The probe of claim 5 where R¹ is methoxy.
 7. The probe of claim 5 where R¹ is methoxy, R²-R⁴ are hydrogen, and x is
 0. 8. The probe of any one of claims 1-7 where the probe is capable of undergoing a condensation/cyclization reaction with a compound comprising a thiol group and an amino group.
 9. The probe of claim 8 where the probe has a first fluorescence spectrum having an emission spectrum maximum at a first wavelength after condensation with the compound, and the probe has a subsequent fluorescence spectrum having an emission spectrum maximum at a second wavelength after cyclization, wherein the first and second wavelengths are different from one another.
 10. The probe of claim 8 where the compound is cysteine, homocysteine, or a combination thereof.
 11. A method for detecting at least one compound comprising a thiol group and an amino group, comprising: combining a sample potentially comprising at least one compound comprising a thiol group and an amino group with a solution comprising a probe having a structure according to general formula II

where R¹-R⁴ independently are hydrogen, hydroxyl, halogen, thiol, thioether, lower aliphatic, or lower alkoxy, x is an integer from 0 to 4, and each R⁵ independently is halogen, hydroxyl, thiol, thioether, lower aliphatic, or lower alkoxy; allowing a reaction between the compound and the probe to proceed for an effective period of time; and detecting the at least one compound by detecting fluorescence of the solution.
 12. The method of claim 11 where the solution has a pH of 7-8.
 13. The method of claim 11 where the at least one compound is cysteine, homocysteine, or a combination thereof.
 14. The method of claim 11 where detecting fluorescence of the solution comprises obtaining a fluorescence spectrum after the effective period of time.
 15. The method of claim 11 where detecting fluorescence of the solution comprises monitoring a fluorescence spectrum of the solution over a period of time ranging from zero minutes to a time greater than or equal to the effective period of time.
 16. The method of claim 11 where R¹ is methoxy, R²-R⁴ are hydrogen, and x is
 0. 17. The method of claim 16 where detecting fluorescence of the solution comprises detecting the fluorescence at 377 nm, at 487 nm, or at 377 nm and 487 nm after the effective period of time.
 18. The method of claim 16 where detecting fluorescence of the solution comprises detecting the fluorescence at 377 nm, at 487 nm, or at 377 nm and 487 nm over a period of time ranging from zero minutes to the effective period of time.
 19. The method of claim 16 where the at least one compound is cysteine and detecting the at least one compound comprises detecting fluorescence of the solution at 487 nm after the effective period of time.
 20. The method of claim 19 where the effective period of time is at least 5 minutes.
 21. The method of claim 19 where the effective period of time is 5-60 minutes.
 22. The method of claim 16 where the at least one compound is homocysteine and detecting the at least one compound comprises detecting fluorescence of the solution at 377 nm after the effective period of time.
 23. The method of claim 22 where the effective period of time is 5-60 minutes.
 24. The method of any one of claims 11-23 where the solution further comprises a surfactant.
 25. The method of claim 24 where the surfactant is cetyltrimethylammonium bromide.
 26. The method of claim 24 where the effective period of time is at least 5 minutes.
 27. The method of claim 24 where R¹ is methoxy, R² is hydrogen, and x is
 0. 28. The method of claim 27 where the effective period of time is 8-10 minutes.
 29. The method of claim 28 where the at least one compound is cysteine and detecting the cysteine comprises detecting fluorescence of the solution at 487 nm after the effective period of time.
 30. The method of claim 28 where the at least one compound is homocysteine and detecting the homocysteine comprises detecting fluorescence of the solution at 377 nm 8-10 minutes after combining the sample and the solution comprising the probe.
 31. The method of claim 28 where the at least one compound is homocysteine and detecting the homocysteine comprises: measuring fluorescence of the solution at 377 nm and 487 nm at a first time 8-10 minutes after combining the sample and the solution comprising the probe; measuring fluorescence of the solution at 377 nm and 487 nm at a second time after combining the sample and the solution comprising the probe, wherein the second time is greater than 8-10 minutes; and determining a difference in fluorescence at each of 377 nm and 487 nm at the first time and the second time, wherein a decrease in fluorescence at 377 nm and a proportional increase in fluorescence at 487 nm indicates presence of homocysteine.
 32. The method of claim 27 where the at least one compound comprises cysteine, homocysteine, glutathione, one or more non-amino thiols, or a combination thereof, the effective period of time is greater than or equal to 9 minutes, and detecting the at least one compound comprises detecting fluorescence of the solution at 377 nm and 487 nm after the effective period of time.
 33. The method of claim 32 further comprising identifying the at least one compound, wherein cysteine is identified based upon stable fluorescence intensity of the solution at 487 nm after 9 minutes and for a subsequent period of time of at least 5 additional minutes, glutathione and/or non-amino thiols are identified based upon stable fluorescence intensity of the solution at 377 nm after 9 minutes and for a subsequent period of time of at least 5 additional minutes, and/or homocysteine is identified based upon proportionally decreasing fluorescence intensity of the solution at 377 nm and increasing fluorescence intensity of the solution at 487 nm after 9 minutes and during a subsequent period of time of at least additional minutes.
 34. A kit for detecting at least one compound comprising a thiol group and an amino group, comprising at least one probe according to general formula II

where R¹-R⁴ independently are hydrogen, hydroxyl, halogen, thiol, thioether, lower aliphatic, or lower alkoxy, x is an integer from 0 to 4, and each R⁵ independently is halogen, hydroxyl, thiol, thioether, lower aliphatic, or lower alkoxy, wherein the probe when combined with a sample comprising at least one compound comprising a thiol group and an amino group for an effective period of time has a different fluorescence emission spectrum than the probe combined with a sample that does not comprise at least one compound comprising a thiol group and an amino group.
 35. The kit of claim 34, where the at least one compound is cysteine, homocysteine, or a combination thereof.
 36. The kit of claim 34, further comprising a buffer solution at physiologic pH.
 37. The kit of claim 36, where the buffer solution is a phosphate solution at pH 7-8.
 38. The kit of claim 36, where the buffer solution further comprises a surfactant.
 39. The kit of claim 38, where the surfactant is cetyltrimethylammonium bromide.
 40. The kit of any one of claims 34-39, further comprising a plurality of disposable containers in which a reaction between the probe and the at least one compound can be performed.
 41. The kit of claim 40, wherein an amount of the probe effective to undergo a detectable change in the probe's fluorescence emission spectrum when reacted with the at least one compound is premeasured into the plurality of disposable containers. 